Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disease caused by mutations in all of the major sarcomeric proteins, including ventricular myosin RLC. Our recent studies have demonstrated that FHC-linked alterations in the Ca2+ binding properties of RLC could be reversed by RLC phosphorylation. Furthermore, our results suggest that a functional coupling that occurs between phosphorylation and Ca2+ binding to RLC during muscle contraction is most likely altered by the FHC mutations. Our preliminary studies on transgenic E22K skinned papillary muscle fibers demonstrated a large decrease in maximal ATPase activity and force per cross-sectional area compared with transgenic WT mouse fibers. Our working hypothesis is that FHC mutations in myosin RLC alter the Ca2+- and or phosphorylation-dependent regulation of cardiac muscle contraction and decrease the level of force/ATPase that in turn may lead to heart failure. To test this hypothesis and to investigate the mechanisms involved in the RLC-linked pathogenesis of FHC, we will study: SPECIFIC AIM 1: EFFECTS OF THE FHC MUTATIONS IN MYOSIN RLC ON THE Ca2+-DEPENDENT REGULATION OF CARDIAC MUSCLE CONTRACTION. Based on our recent results with transgenic E22K mouse model and the results of profoundly decreased ATPase and force in the N47K- and R58Q-reconstituted fiber systems, it is predicted that the Ca2+ regulation of force/ATPase in intact and skinned papillary muscle fibers derived from N47K and/or R58Q transgenic mice will be even more altered compared to non-transgenic, transgenic-WT or A13T mice. Specifically these transgenic mouse lines will be examined for: a) Ca2+-sensitivity and maximal levels of force and actomyosin ATPase; b) alterations in energy cost or rate of cross-bridge dissociation (ATPase/force) c) kinetics of force development/relaxation (ktr and caged Ca-chelator); d) velocity of shortening; e) diastolic and systolic [Ca2+] and force; f) duration of [Ca2+] and force transients; g) the ability of the muscle to do the work against a constant afterload. SPECIFIC AIM 2: PHYSIOLOGICAL CONSEQUENCES OF THE FHC RLC MUTATIONS ON THE PHOSPHORYLATION-DEPENDENT REGULATION OF CARDIAC MUSCLE CONTRACTION. Studies utilizing various animal models have shown a correlation between the level of RLC phosphorylation and cardiac performance. We hypothesize that FHC mutations interfere with the phosphorylation-dependent regulatory function of the RLC during muscle contraction. We will study the effects of RLC phosphorylation and the physiological significance of phosphorylation in the pathological heart in these transgenic FHC RLC mice. These studies will correlate the observed effects of the RLC mutations in the proposed animal models with the pathogenesis of FHC in humans and will decipher the key mechanisms of the RLC-linked FHC.